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Oligo(ethylene glycol)-functionalized Squaraine Fluorophore as NIR Probe for In-vivo Fluorescent Detection of Diagnostic Enzyme Bo Wu, Yi Lin, Bowen Li, Chenyue Zhan, Fang Zeng, and Shuizhu Wu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01968 • Publication Date (Web): 13 Jul 2018 Downloaded from http://pubs.acs.org on July 14, 2018

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Analytical Chemistry

Oligo(ethylene glycol)-functionalized Squaraine Fluorophore as NIR Probe for In-vivo Fluorescent Detection of Diagnostic Enzyme Bo Wu‡, Yi Lin‡, Bowen Li, Chenyue Zhan, Fang Zeng*, Shuizhu Wu* State Key Laboratory of Luminescent Materials and Devices, College of Materials Science and Engineering, South China University of Technology, Guangzhou 510640, China. ABSTRACT: Squaraine dyes have excellent photostability with intensive absorption and strong fluorescence in the near infrared (NIR) region. However, they display a strong tendency to aggregate in aqueous media due to their poor water solubility, often causing fluorescence quenching that severely limits their in-vivo applications especially for detecting/imaging diagnostic enzymes. In this work, an oligo(ethylene glycol)-functionalized squaraine fluorophore has been developed as a NIR fluorescent probe, which can detect/image the activities of diagnostic enzyme (leucine aminopeptidase) both in vitro and in vivo. The probe shows near infrared absorption and emission, low detection limit (0.61 ng / mL), relatively good aqueous solubility, high selectivity and little toxicity. In addition, the probe herein was successfully used to tracking endogenous leucine aminopeptidase both in vitro and in vivo with a nude mouse model.

Fluorescence detection/imaging technique is an effective tool for monitoring biological targets and processes and providing outstanding spatio-temporal resolution at the molecular level with a high sensitivity.1-11 For non-invasive in-vivo detection/imaging, fluorescent probes with absorption and emission in the NIR (near infrared) region (650–900 nm) are particularly advantageous and highly sought-after because of the low absorption of biological molecules, little photodamage to biological samples, relatively deep tissue penetration and low background interference from autofluorescence of biomolecules in the living systems.16-23 Squaraine dyes show intensive absorption and strong emission typically in NIR region24-28 and have been successfully applied in solar cells due to their good optical properties and excellent photostability;29-31 hence these dyes can be designed as promising NIR fluorescent probes for detecting biomolecules. However, squaraine dyes display a strong tendency to aggregate in aqueous solution partly due to their poor water solubility, often causing fluorescence quenching due to aggregation-caused quenching (ACQ)32-34 property thus limiting its in-vivo applications. Due to these properties, many squarainebased fluorescent detection systems are often utilized for metal ion recognition and protein detection in vitro via aggregation/ disaggregation or coordination process.35-38 Furthermore, many enzymes are very important biomarkers for disease diagnosis, yet only a few squaraine fluorophores have been used for detecting/imaging diagnostic enzymes in vitro.39-41 On the other hand, it is well known that incorporation of hydrophilic poly(ethylene glycol) (PEG) and oligo(ethylene glycol) (OEG) onto drugs can enhance their water solubility and improve biocompatibility via minimizing nonspecific interaction with biological molecules such as proteins, RNA and DNA, and achieve prolonged circulation time.42-45 OEG with a welldefined length can easily undergo chemical modification

which is preferred for functionalization. We envision that, incorporating an OEG (e.g. triethylene glycol) on one side and a hydrophilic substrate moiety on the other side of a squaraine scaffold may ameliorate the above limitation and thus generate NIR fluorescent probe for detecting diagnostic enzyme. Toward this end, by employing leucine aminopeptidase (LAP) as a model diagnostic enzyme we constructed an OEGfunctionalized squaraine NIR probe (USSQ-Leu) for in-vitro and in-vivo LAP detection and imaging. This enzyme is normally found in liver cells and small intestine cells; and abnormal expression of LAP is closely related to several human diseases including hepatitis46-48 and liver cancer49-51, enabling LAP as an important biomarker (diagnostic enzyme) for detecting these diseases. The probe herein with NIR absorption and emission has quite good aqueous solubility, and shows sensitive, rapid and selective response toward LAP. Moreover, this probe has been successfully used in imaging HepG2 xenograft tumor in a mouse model. As shown in Scheme 1, this probe contains three parts: an unsymmetrical squaraine (USSQ) unit serves as the NIR fluorescence reporter, the hydrophilic L-leucine moiety serves as the enzyme substrate (the recognition moiety for LAP) and the fluorescence quencher, and an OEG (triethylene glycol) moiety for improving aqueous solubility and biocompatibility. The probe USSQ-Leu was synthesized by coupling the L-leucinecontaining quinolone and the OEG-containing indole with squaric acid as shown in Scheme S1. For comparison, two other squaraine-based fluorophores (namely one with no OEG group, SQ-0; and the other with two OEG groups, SQ-2) were also synthesized as shown in Scheme S2. And the structures of the intermediates and the final product were confirmed by 1H NMR and HR-MS spectrometry (Figures S1–16).

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NMR spectrometer. Chemical shifts are reported in ppm value using the peak of residual proton signals of TMS as the internal reference. High-resolution mass spectra (HRMS) were obtained with AB Sciex Triple TOF 5600+ mass spectrometer. Absorption spectra were obtained with Hitachi U-3010 UV-vis spectrophotometer. Hitachi F-4600 fluorescence spectrophotometer was used for measuring fluorescence spectra. Olympus IX 71 with a DP72 color CCD was used for obtaining fluorescence images. In vivo imaging was performed on Ami small animal imaging system (SI Imaging). Spectroscopic Measurement. Stock solution of USSQ-Leu (1.0 mM) in DMSO was made and used upon dilution with Phosphate buffered water (referred to as PBS, pH 7.4, 10 mM). The stock solutions (10.0 mM or 200 U/L) of CaCl2, MgCl2, ZnCl2 and other bioanalytes (FeCl2, FeCl3, CuCl2, CaCl2, GSH, Cys, Leu, Gly, Arg, Phe, NaOCl, H2O2, Lactase, DT-diaphorase, carboxylesterase, nitroreductase, LAP) were prepared in PBS (10 mM, pH 7.4). For optical measurement was conducted with similar procedures as previously reported33 and briefly described as follows: the solution of USSQ-Leu was incubated with the analyte (LAP or others) in PBS (pH 7.4, 10 mM, containing 10% DMSO) under certain pH in standard quartz cuvette of optical path length of 1 cm under 37 °C (the final USSQ-Leu concentration was 5 µM). The optical spectrum of the above solution was obtained after being measured at an indicated procedure. For fluorescence measurements, excitation wavelength was 650 nm with slit width of 5 nm × 10 nm. For the selectivity experiment, USSQ-Leu was incubated respectively with every analyte for 30 min, the fluorescent intensity at 710 nm was then measured. In order to compare the change of fluorescent intensity, we used the fluorescent intensity at 710 nm of the USSQ-Leu (5 µM) in PBS (pH 7.4, 10 mM, containing 10% DMSO) as a standard value, defined as F0.

Scheme 1. Schematic overview of the sensing mechanism for detecting LAP in vitro and in vivo via enzymatic reaction by the probe USSQ−Leu.

EXPERIMENTAL SECTION Materials and General Instrumentation. 4-Nitroaniline, crotonaldehyde, stannous chloride dihydrate, BOC-L-Leucine, PyBop, N,N-Diisopropylethylamine (DIPEA), ethyl iodide, triethylene glycol monomethyl ether, p-toluene sulfonyl chloride, 2,3,3-trimethylindolenine, 1,2,3,3-tetramethyl-3Hindolium iodide and squaric acid were purchased from SigmaAldrich Reagents without further purification. DMEM was purchased from KeyGen BioTECH. The solvents including carbon tetrachloride, acetone, dichloromethane (DCM) and N,N-dimethylformamide (DMF), etanol, n-butyl alcohol, tolune were dried with 5A molecular sieve and distilled under nitrogen atmosphere. Other solvents used in this study were of analytical grade and used as received. In the experiments water was triple-distilled. Column chromatography was conducted with silica gel (300-400 mesh). pH value was measured with Sartorius PB-10 pH meter. Synthesis of USSQ-Leu (Compound 10, the probe). The compound 9 (1 equiv., 0.25 mmol, 100 mg) was dissolved in a toluene/n-butanol mixture (1/1, 40 mL) and compound 4 (1 equiv., 0.25 mmol, 130 mg) was added. The flask was equipped with a Dean-Stark apparatus and the solution was heated at 125 °C overnight. The solvents were evaporated and the crude product was purified by column chromatography (CH2Cl2: CH3OH = 50 : 3 v/v) to obtain a blue solid as USSQBocLeu. Next, the blue solid was dissolved in 10 mL CH2Cl2 and 1 mL TFA was added. The reaction solution was stirred under room temperature for 3 h, then the solvents were evaporated and the product 10 was obtained (40 mg, 15%). 1H NMR (600 MHz, DMSO-d6) δ 9.45 (d, J = 9.3 Hz, 1H), 8.27 (d, J = 2.3 Hz, 1H), 8.07 (d, J = 9.4 Hz, 1H), 7.98 (d, J = 9.5 Hz, 1H), 7.95 (dd, J = 9.3, 2.3 Hz, 1H), 7.39 (d, J = 7.2 Hz, 1H), 7.27 – 7.22 (m, 1H), 7.12 (d, J = 7.9 Hz, 1H), 7.01 (t, J = 7.4 Hz, 1H), 5.90 (s, 1H), 5.61 (s, 1H), 4.55 (d, J = 5.9 Hz, 2H), 4.09 (s, 2H), 3.74 (t, J = 5.6 Hz, 2H), 3.54 – 3.51 (m, 2H), 3.47 – 3.44 (m, 2H), 3.45 – 3.41 (m, 2H), 3.36 – 3.34 (m, 2H), 3.18 (s, 3H), 1.80 – 1.74 (m, 1H), 1.66 (s, 6H), 1.55 – 1.52 (m, 1H), 1.44 (t, J = 7.2 Hz, 3H), 0.92 (dd, J = 14.3, 6.6 Hz, 6H), 0.86 (t, J = 7.0 Hz, 2H). HR-MS (ESI): calculated for C40H50N4O6 ([M]+) 682.3730, found: 682.3729. Measurements. The instruments for measurements were as previously reported33 and briefly described as follows: 1H NMR spectra were obtained with a Bruker Avance 600 MHz

Water solubility Determination. In a centrifugal tube an oversaturated aqueous solution was prepared in ultrapure water. Then the process of centrifugation (30 min at 9000 r/min and 25°C) for the solution and collecting the supernatant at 25°C was performed to get 10 mL of saturated solution. After that, the saturated solution was undergoing freeze-drying over night to obtain the weight of the solute. The solubility was finally determined by using the volume (10 mL) and the weight of solute. Cell imaging. HepG2 cells, LO2 cells and L929 cells were cultured in DMEM that was added with 10% FBS for 24 hours incubation. Then cells were seeded in a 6-well plate. Cells were allowed to grow to 50–70% confluence and washed with DMEM. And the cells were incubated with the USSQ-Leu solution (containing 1% DMSO) in a humidified incubator (37 °C, 5% CO2) for 2 hours directly or after the following treatments: For Ace (acetamidophenol) stimulation, Ace (1 mM) solution was added to the 6-well plates containing adherent cells in DMEM supplemented with 10% FBS in a humidified incubator (37 °C, 5% CO2), before the probe was added, and the cells were incubated for 24 hours. For bestatin inhibition, bestatin solution was pretreated to the 6-well plates containing adherent cells in in DMEM supplemented with 10% FBS in a humidified incu-


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Analytical Chemistry USSQ-Leu in the presence of LAP were recorded as well. As expected (Figure 1a), as LAP level was increased (0 – 40 U/L) and upon incubation with USSQ-Leu (5 µM) for 30 minutes under physiological conditions, the fluorescence intensity gradually increased, while when further LAP (> 40 U/L) was added, no more significant change could be noticed. Then the time-dependent fluorescence spectra of the USSQ-Leu with 40 U/L of LAP in PBS was also measured, the fluorescence intensity at 710 nm increased strikingly and reached the plateau in 30 minutes (Figure 1b). Moreover, in order to further confirm the enzymatic cleavage reaction by LAP has occurred, the reaction solution was analyzed by HR-MS (Figure 1c), after treatment with LAP for 10 min, the mass peak at 570.2962 corresponding to the reaction product USSQ-NH2 could be observed. And the enzymatic cleavage reaction was also confirmed by HPLC. As illustrated in Figure S19, USSQLeu and the reaction product can be identified at 3.02 min and 1.53 min in HPLC chromatogram. In addition, the apparent kinetic parameter of the USSQ-Leu (the probe) was deter54 mined in accordance with Michaelis-Menten equation , and correspondingly the Michaelis constant (Km) value was 10.8 µM (Figure S20).

bator (37 °C, 5% CO2), before the probe was added, the cells were incubated for 1 hour. Afterwards, the 6-well plates were washed with PBS for three times. By using the Olympus IX71 inverted fluorescence microscope equipped with a DP72 color CCD, fluorescence images were obtained. Cell viability assay. Cells (5000 cells/well) were seeded in 96-well plates and incubated with the probe USSQ-Leu with varied concentrations (from 0 to 100 µM, containing 1% DMSO) at 37 °C. The cells without the treatment were used as control. After incubation for 24 hours, the wells were washed three times with PBS and treated with DMEM medium containing 0.5 mg/mL MTT for another 4 hours. The resulting formazan crystal was dissolved in 150 µL of DMSO (after removing the medium carefully) and then the absorbance was recorded (at 570 nm). The cell viability assays were performed using a Thermo MK3 ELISA plate reader. The three independent experiments performed in six replicates were used to obtain the statistical mean and standard deviation. In vivo fluorescent imaging. The experiments involving animals were carried out in Laboratory Animal Center of South China Agricultural University (SCAU), and the experimental protocols have been approved by the Animal Ethics Committee of SCAU in accordance with the guidelines for the care and use of laboratory animals. The 5-6-week-old BALB/c nude mice were supplied by Guangdong Medical Laboratory Animal Center and maintained under standard conditions. For in vivo imaging, the nude mice were subcutaneously injected with 100 µL HepG2 cells (5 × 106 cells per mouse) on the right oxter to establish tumor model. The tumor-bearing mice were randomly divided into 2 treatment groups for earlyand advanced-stage of cancer (n = 6/group). Tumors were allowed to grow to a volume of 25-30 (early-stage, around 5 days upon inoculation of HepG2 cells) mm3 and around 500 (advanced-stage, around 21 days upon inoculation of HepG2 cells) mm3 and then used for experiment. And control group (n = 6) was established with no cancer cells injected. To detect the endogenous LAP activity, the probe USSQLeu (50 µM, 50 µL) was injected through tail vein. The in vivo imaging was obtained by using an AMI small animal imaging system of SI Imaging Co. with λex/em = 650/710 nm. In nude mice xenograft tumor model, the early-stage tumor usually has a volume of around 60 mm3 or smaller, and the advanced-stage tumor has a volume of around 500 mm3 or larger. 52,53 Ex vivo fluorescent imaging. The tumor-bearing mice with different stages of tumor were intravenously injected with the probe USSQ-Leu (50 µM, 50 µL). After 4 hours, the injected mice were sacrificed with CO2, and the tumor and other organs were dissected and imaged with λex/em = 650/710 nm.

Figure 1. (a) Concentration-dependent fluorescence spectra of the USSQ-Leu toward LAP (the concentration of LAP is from 0 to 100 U/L, reaction at 37 °C for 30 min in PBS with 10% DMSO, pH = 7.4), inset: fluorescence intensity at 710 nm versus LAP concentration; (b) Time-dependent fluorescence spectra of the USSQ-Leu (5 µM) incubated with 40 U/L LAP (PBS with 10% DMSO, pH = 7.4 at 37 °C), inset: fluorescence intensity at 710 nm versus incubating time; λex = 650 nm. (c) HR-MS spectrum of the reaction solution of the USSQ-Leu (5 µM) incubated for 10 min with LAP (40 U/L). F0 represents the emission intensity at 710 nm of the USSQLeu (5 µM) in PBS (pH 7.4, 10 mM, containing 10% DMSO).

RESULTS AND DISCUSSION Spectral Properties of USSQ-Leu with or without LAP. The probe USSQ-Leu exhibits a strong absorption peak at around 650 nm and weak fluorescence, as shown in Figure S17. However, after incubation with LAP under physiological condition, a remarkable fluorescence enhancement (9-fold) at 710 nm occurs (Figure S18). The fluorescence spectra of the

Aqueous solubility of the probe. To evaluate effects of OEG moiety on the water solubility of the probe, we also synthesized the other two squaraine fluorophores SQ-0 and SQ-2 which contain zero or two OEG group(s) respectively (the



details of the synthesis and related characterizations are shown in Scheme S2 and Figures S15 and S16 in the Supporting Information). Their water solubility was determined to be 0.0212 mg (SQ-0), 0.341 mg (the probe USSQ-Leu) and 1.278 mg (SQ-2). And the absorption spectra of these three squaraine fluorophores (with same concentration, 5 µM) in the solutions containing different ratio of phosphate-buffered water (PBS) and DMSO were measured. As displayed in Figure 2, we can find out that the spectra of SQ-0 changed significantly when the ratio of PBS was raised, probably due to the fact that with higher amount of PBS, the fluorophore SQ-0 could not completely dissolve even in such dilute solution (5 µM). While the spectra of the probe and SQ-2 were relatively stable, which further confirmed that the introduction of OEG improved the aqueous solubility of squaraine fluorophores.

Leu with some other bioanalytes such as CaCl2, MgCl2, ZnCl2 and other bioanalytes (FeCl2, FeCl3, CuCl2, CaCl2, GSH, Cys, Leu, Gly, Arg, Phe, NaOCl, H2O2, Lactase, DT-diaphorase, carboxylesterase, nitroreductase, gamma-glutamyl transferase, prolidase, methionine aminopeptidase) were also investigated, including some biologically important metal ions, biomolecules, and other enzyme species. As displayed in Figure 3c, the emission intensity at 710 nm had negligible variation in the presence of possible competitive analytes. But after the probe was treated with LAP, the emission intensity displayed a striking enhancement. The results reveal that the probe USSQ-Leu is a quite specific probe towards LAP in the biological environment. Moreover, we also performed the control experiments by employing LAP inhibitor bestatin53-58. As shown in Figure S21, the LAP inhibitor led to much weaker fluorescence as a result of less LAP-induced enzymatic reaction, which further confirms that the change of the fluorescence signal is triggered by LAP.

Figure 2. (a) Chemical structures of squaraine fluorophores. Absorption spectra of (b) SQ-0, (c) the probe USSQ-Leu, (d) SQ-2 in the solutions with different ratio of PBS and DMSO (Concentration = 5µM).

Effects of pH. To confirm the probe USSQ-Leu can be utilized for bioimaging, the effects of pH on the probe’s response to LAP was also explored. As shown in Figure 3a, the fluorescence of USSQ-Leu was almost unaffected by the variation of pH. However, when pretreated with LAP for 30 min, the USSQ-Leu solution exhibited recognizable fluorescence enhancement in the pH range of 4.0 - 10.0. Markedly, the emission intensity of USSQ-Leu in the presence of LAP reached the maximum when pH was around 7.0 and it varied slightly under the pH ranging from 4.0 to 10.0, which indicates that the probe would be applicable in biological milieu. Photostability of the probe USSQ-Leu. To prove the probe has high photostability, the fluorescence intensity of the probe and the probe pretreated with LAP under the irradiation of xenon lamp (150 W) were recorded. As displayed in Figure 3b, the fluorescence of USSQ-Leu showed nearly no change upon UV light irradiation. Meanwhile, when pretreated with LAP for 30 min, the USSQ-Leu solution exhibited very good photostabily as well. Selectivity of the probe USSQ-Leu. Given that intracellular environment was of complex biological system, before the cellular imaging experiment, the performance of the USSQ-

Figure 3. (a) Effects of pH on the fluorescence of the USSQ-Leu (5 µM) and the USSQ-NH2 (5 µM of USSQ-Leu treated with 40 U/L of LAP in PBS (pH 7.4, 10 mM, containing 10% DMSO) at 37 °C for 30 min before PH change). λex/em = 650/710 nm; (b) Photostability of the USSQ-Leu (5 µM) and the USSQ-NH2 (5 µM of USSQ-Leu treated with 40 U/L of LAP in PBS (pH 7.4, 10 mM, containing 10% DMSO) at 37 °C for 30 min before irradiation) under the irradiation of xenon lamp (150 W); (c) Fluorescence intensity for the probe (5 µM) at 710 nm in PBS (pH = 7.4, 10 mM, PBS with 10% DMSO) in the presence of various potential species for 30 min: (1) control; (2) KCl (150 mM); (3) CaCl2 (5 mM); (4) MgCl2 (5 mM); (5) ZnCl2 (100 µM); (6) FeCl2 (1 mM); (7) FeCl3 (1 mM); (8) CuCl2 (1 mM); (9) CaCl2 (1 mM); (10) GSH (5 mM); (11) Cys (5 mM); (12) Leu (5 mM); (13) Gly (5 mM); (14) Arg (5 mM); (15) Phe (5 mM); (16) NaOCl (100 µM) ; (17) H2O2 (100 µM); (18) Lactase (100 U/L); (19) DTdiaphorase (100 U/L); (20) carboxylesterase (100 U/L); (21) nitroreductase (100 U/L); (22) gamma-glutamyl transferase (100 U/L); (23) prolidase (100 U/L); (24) methionine aminopeptidase (100 U/L); (25) LAP (40 U/L). λex/em = 650/710 nm. F0 represents the fluorescent intensity at 710 nm of the USSQ-Leu (5 µM) in PBS (pH 7.4, 10 mM, containing 10% DMSO).


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Sensitivity of the probe USSQ-Leu for LAP. To investigate the USSQ-Leu’s fluorescence turn-on response to LAP with different concentrations, LAP (0 – 15 U/L) were added to USSQ-Leu solution (5 µM) in PBS (pH 7.4, 10 mM, containing 10% DMSO) at 37 °C for 30 min, and then the fluorescent turn-on response at 710 nm was recorded. The result was displayed in Figure S22, and the limit of detection for LAP was determined as 0.61 ng / mL. Compared various studies, the USSQ-Leu is a quite good probe for dection of LAP.59-62 Fluorescence Imaging in Living Cells. Subsequently, we investigated the ability of the USSQ-Leu for detecting LAP inside cells. As for the cell-imaging experiments, we employed DMSO as co-solvent, because it is the most commonly-used organic solvent that has a high water affinity and low systemic toxicity, and also it is widely used in several procedures of cell-culturing experiment including cell freezing and cell thawing.63 First of all, we explored the influence of the USSQ-Leu on the viabilities of the HepG2 cells and the cellular uptake of the USSQ-Leu. The viability of HepG2 cells upon treatment with the USSQ-Leu were assessed using MTT assays. As shown in Figure S23, the results showed that there is insignificant cytotoxicity when the HepG2 cells were incubated with 0 – 100 µM of the USSQ-Leu for 24 hours. Afterwards, the cell uptake was also evaluated by flow cytometry, and the mean fluorescence intensities showed a significant increment when the USSQ-Leu was incubated with HepG2 cells compared to the cells which were incubated without the probe for 2 hours (Figure S24), indicating the probe molecules can be efficiently internalized by cells. Since the probe shows good biocompatibility and excellent cellular uptake, we then investigated its ability of detecting endogenous LAP in three kinds of cell lines which expressed LAP differently. As shown in Figure 4, as the LAP-overexpressed HepG2 cells were incubated with the USSQ-Leu, it shows a bright red image (Figure 4a). While the fluorescence is much less bright in LO2 cells (Figure 4d) which expressed less LAP. To further confirm that the USSQ-Leu can be activated by endogenous LAP, we then used acetaminophen64,65 (Ace) which can upregulate the LAP level by causing damage to liver cells and bestatin (a specific inhibitor of LAP to pretreat the cells) in the cellular imaging experiments. As shown in Figure 4e, the Acepretreated LO2 cells show strong red fluorescent imaging compared to the normal LO2 cells. While the fluorescence of the cells which are treated with bestatin (Figure 4b and 4f) look almost the same as that of the untreated LO2 cells. Moreover, we chose L929 cells which contain nearly no LAP as the control and incubated them with the probe, and it turns out to be almost dark (Figure 4c). The above results verify the outstanding ability of the probe USSQ-Leu for fluorescent sensing of LAP in cells and confirm that our probe could serve as a detection system for tracking LAP in vitro.

Figure 4. Fluorescent and bright field images of cells. (a and a’) HepG2 cells incubated with the probe USSQ-Leu (5 µM) for 2 h; (b and b’) HepG2 cells pretreated with bestatin (50 µM) for 1 h and then incubated with probe (5 µM) for 2 h; (c and c’) L929 cells incubated with the probe (5 µM) for 2 h; (d and d’) LO2 cells incubated with the probe (5 µM) for 2 h; (e and e’) LO2 cells pretreated with Ace for 24 h (1 mM) and then treated with the probe for 2 h (5 µM); (f and f’) LO2 cells pretreated with bestatin (10 µM) for 1 h and then incubated with the probe (5 µM) for 2 h.

Fluorescence Imaging in vivo. Since the probe can be used for monitoring LAP activity under in-vitro condition, plus its NIR properties, we thereby use it to detect xenograft tumor in BALB/c nude mice by imaging the overexpressed LAP. The mice were subcutaneously injected with ca. 5×106 HepG2 cells in right oxter. As shown in Figure 5a, for the advanced-stage HepG2 xenograft tumor model (the tumors were around 500 mm3 in volume), after the HepG2 tumor-bearing mice were intravenously injected with 50 µL of USSQ-Leu, a weak fluorescence could be observed in the tumour region in 30 min on an AMI small animal imaging system (SI Imaging Co.), and the fluorescence gradually increased and remained intense until 4 hours, then the fluorescence gradually decreased. Moreover, after the fluorescence in the abdominal cavity disappeared entirely, we could still observe a relatively recognizable fluorescence at the tumor site. In addition, we used the



healthy mice intravenously injected with PBS (as the control group). Organs of panels (b) and (c) were dissected from the tumor-bearing mice 4 hours after being intravenously injected with the probe for different stages of HepG2 xenograft tumor.

USSQ-Leu to image the LAP activity in the early-stage HepG2 xenograft tumor (the tumors were around 25-30 mm3 in volume) in vivo. As depicted in Figure 5b, when the mice were intravenously injected with 50 µL of probe, a significant fluorescence enhancement was observed in 4 hours at the tumor site and remained this way till 24 h. The above results indicate that the USSQ-Leu is capable of imaging the LAPoverexpressed tumors in vivo. Moreover, strong fluorescence also appeared in livers mainly causing by the metabolism of the activated probe and high level of LAP due to liver damage as a side-effect of cancer.

CONCLUSION In summary, we have successfully developed a squarainebased NIR probe for detecting LAP via enzymatic reaction. The probe shows improved aqueous solubility, high sensitivity and selectivity. By exploiting these advantageous properties, we have successfully used the probe to detect the activity of LAP in vitro, and image the LAP-overexpressed tumors in vivo. The probe herein may be applicable in tracking and positioning hepatoma where LAP is over-expressed, and the approach for constructing the probe may offer helpful insights for designing other squaraine-based fluorophores as useful NIR bioprobes for applications in complicated physiological environment.

ASSOCIATED CONTENT Supporting Information

Figure 5. Typical fluorescent images of the (a) advanced-stage or (b) early-stage HepG2 xenograft tumor model mice after being injected with the USSQ-Leu for 0-48 hours.

Additional information includes the synthesis, 1H NMR spectra, mass spectra, absorption spectra, fluorescence spectra, HPLC chromatogram, lineweaver-burk plot, bestatin experiment, the detection limit, cell viability and cellular uptake. The Supporting Information is available free of charge on the ACS Publications website.

Fluorescence Imaging ex vivo. Furthermore, the mice were sacrificed humanely and some organs were dissected for fluorescent imaging (shown in Figure 6). As for the control group (healthy mice without injection of the probe), nearly no fluorescence could be observed (Figure 6a). In Figure 6b (early stage) and 6c (advanced stage), strong fluorescence was found in tumors and livers which also contained a high level of LAP. And the kidneys were also found to exhibit weak fluorescence probably because part of the probe molecules could be metabolized through kidney. Taken together, the results indicate that the probe herein can serve as an efficacious probe for LAP in vivo.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] *E-mail:[email protected]

Author Contributions ‡These authors contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We acknowledge the financial support by NSFC (51673066, 21574044 and 21474031), the Science and Technology Planning Project of Guangzhou (Project No. 201607020015) and the Natural Science Foundation of Guangdong Province (2016A030312002)

REFERENCES (1) Chang, H.-C.; Ho, J. A. Gold Nanocluster-assisted Fluorescent Detection for Hydrogen Peroxide and Cholesterol Based on the Inner Filter Effect of Gold Nanoparticles. Anal. Chem. 2015, 87, 1036210367. (2) Lauber, M. A.; Yu, Y. Q.; Brousmiche, D. W.; Hua, Z.; Koza, S. M.; Magnelli, P.; Guthrie, E.; Taron, C. H.; Fountain, K. J. Rapid Preparation of Released N-Glycans for HILIC Analysis Using a Labeling Reagent that Facilitates Sensitive Fluorescence and ESI-MS Detection. Anal. Chem. 2015, 87, 5401-5409.

Figure 6. Typical Images of dissected organs from (a) control mice; (b) early-stage tumor-bearing mice; (c) advanced-stage tumor-bearing mice. Organs of panel (a) were dissected from the


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Analytical Chemistry (3) Li, G.; Fu, H.; Chen, X.; Gong, P.; Chen, G.; Xia, L.; Wang, H.; You, J.; Wu, Y. Facile and Sensitive Fluorescence Sensing of Alkaline Phosphatase Activity with Photoluminescent Carbon Dots based on Inner Filter Effect. Anal. Chem. 2016, 88, 2720-2726. (4) Teng, Y.; Jia, X.; Li, J.; Wang, E. Ratiometric Fluorescence Detection of Tyrosinase Activity and Dopamine Using ThiolateProtected Gold Nanoclusters. Anal. Chem. 2015, 87, 4897-4902. (5) Zhang, L.; Han, Y.; Zhu, J.; Zhai, Y.; Dong, S. Simple and Sensitive Fluorescent and Electrochemical Trinitrotoluene Sensors Based on Aqueous Carbon Dots. Anal. Chem. 2015, 87, 2033-2036. (6) Karpenko, I. A.; Collot, M.; Richert, L.; Valencia, C.; Villa, P.; Mély, Y.; Hibert, M.; Bonnet, D.; Klymchenko, A. S. Fluorogenic Squaraine Dimers with Polarity-Sensitive Folding as Bright Far-Red Probes for Background-Free Bioimaging. J. Am. Chem. Soc. 2014, 137, 405-412. (7) Liu, W.; Liu, S.-J.; Kuang, Y. Q.; Luo, F. Y.; Jiang, J. H. Developing Activity Localization Fluorescence Peptide Probe Using Thiol-Ene Click Reaction for Spatially Resolved Imaging of Caspase8 In Live Cells. Anal. Chem. 2016, 88, 7867-7872. (8) Hang, Y.; Wang, J.; Jiang, T.; Lu, N.; Hua, J. Diketopyrrolopyrrole-Based Ratiometric/Turn-On Fluorescent Chemosensors for Citrate Detection in The Near-Infrared Region by an Aggregation-Induced Emission Mechanism. Anal. Chem. 2016, 88, 1696-1703. (9) Guo, F.; Gai, W. P.; Hong, Y.; Tang, B. Z.; Qin, J.; Tang, Y. Aggregation-Induced Emission Fluorogens as Biomarkers to Assess the Viability of Microalgae in Aquatic Ecosystems. Chem. Commun. 2015, 51, 17257-17260. (10) Li, M.; Liu, Z.; Wang, S.; Calatayud, D. G.; Zhu, W. H.; James, T. D.; Wang, L.; Mao, B.; Xiao, H. N. Fluorescence Detection and Removal of Copper from Water Using a Biobased and Biodegradable 2D Soft Material. Chem. Commun. 2018, 54, 184-187. (11) Li, B.; Xie, X.; Chen, Z.; Zhan, C.; Zeng, F.; Wu, S. Tumor Inhibition Achieved by Targeting and Regulating Multiple Key Elements in EGFR Signaling Pathway Using a Self‐Assembled Nanoprodrug. Adv. Funct. Mater. 2018, 28, 1800692. (12) Guo, Z.; Park, S.; Yoon, J.; Shin, I. Recent Progress in the Development of Near-Infrared Fluorescent Probes for Bioimaging Applications, Chem. Soc. Rev. 2014, 43, 16-29. (13) Lim, S. Y.; Hong, K. H.; Kim, D. I.; Kwon, H.; Kim, H. J. Tunable Heptamethine-Azo Dye Conjugate as an NIR Fluorescent Probe for the Selective Detection of Mitochondrial Glutathione over Cysteine and Homocysteine. J. Am. Chem. Soc. 2014, 136, 70187025. (14) Zheng, F.; Guo, S.; Zeng, F.; Li, J.; Wu, S. Ratiometric Fluorescent Probe for Alkaline Phosphatase Based on BetaineModified Polyethylenimine via Excimer/Monomer Conversion. Anal. Chem. 2014, 86, 9873-9879. (15) Chiu, C. F.; Saidi, W. A.; Kagan, V. E.; Star, A. DefectInduced Near-Infrared Photoluminescence of Single-Walled Carbon Nanotubes Treated with Polyunsaturated Fatty Acids. J. Am. Chem. Soc. 2017, 139, 4859-4865. (16) Wang, X.; Dai, Y.; Liu, R.; He, X.; Li, S.; Wang, Z. L. LightTriggered Pyroelectric Nanogenerator Based on a Pn-Junction For Self-Powered Near-Infrared Photosensing. ACS nano 2017, 11, 83398345. (17) Li, B.; Liu, P.; Wu, H.; Xie, X.; Chen, Z.; Zeng, F.; Wu, S. A Bioorthogonal Nanosystem for Imaging and In Vivo Tumor Inhibition. Biomaterials, 2017, 138, 57-68. (18) Wu, Y.; Wang, J.; Zeng, F.; Huang, S.; Huang, J.; Xie, H.; Yu, C.; Wu, S. Pyrene Derivative Emitting Red or Near-Infrared Light with Monomer/Excimer Conversion and Its Application to Ratiometric Detection of Hypochlorite. ACS Appl. Mater. Interfaces 2016, 8, 1511-1519. (19) Kong, F.; Liang, Z.; Luan, D.; Liu, X.; Xu, K.; Tang, B. A Glutathione (GSH)-Responsive Near-Infrared (NIR) Theranostic Prodrug for Cancer Therapy and Imaging. Anal. Chem. 2016, 88, 6450-6456.

(20) Hu, Q. H.; Yu, C. M.; Xia, X. T.; Zeng, F.; Wu, S. A Fluorescent Probe for Simultaneous Discrimination of GSH and Cys/Hcy in Human Serum Samples via Distinctly-Separated Emissions with Independent Excitations. Biosens. Bioelectron. 2016, 81, 341-348. (21) Yuan, S.; Wang, F.; Yang, G.; Lu, C.; Nie, J.; Chen, Z.; Ren, J.; Qiu, Y.; Sun, Q.; Zhao, C.; Zhu, W. H. Highly Sensitive Ratiometric Self-Assembled Micellar Nanoprobe for Nitroxyl and Its Application In Vivo. Anal. Chem. 2018, 90, 3914-3919. (22) Wang, F.; Zhou, L.; Zhao, C.; Wang, R.; Fei, Q.; Luo, S.; Guo, Z.; Tian, H.; Zhu, W. H. A Dual-response BODIPY-based Fluorescent Probe for the Discrimination of Glutathione from Cystein and Homocystein. Chem. Sci. 2015, 6, 2584-2589. (23) Gu, K.; Liu, Y.; Guo, Z.; Lian, C.; Yan, C.; Shi, P.; Tian, H.; Zhu, W. H. In Situ Ratiometric Quantitative Tracing of Intracellular Leucine Aminopeptidase Activity via an Activatable Near-Infrared Fluorescent Probe. ACS appl. Mater. Interfaces 2016, 8, 2662226629. (24) Jin, B.; Zhang, X.; Zheng, W.; Liu, X.; Zhou, J.; Zhang, N.; Wang, F.; Shangguan, D. Dicyanomethylene-Functionalized Squaraine as a Highly Selective Probe for Parallel G-Quadruplexes. Anal. Chem. 2014, 86, 7063-7070. (25) Qin, C.; Numata, Y.; Zhang, S.; Yang, X.; Islam, A.; Zhang, K.; Chen, H.; Han, L. Novel Near‐Infrared Squaraine Sensitizers for Stable and Efficient Dye‐Sensitized Solar Cells. Adv. Funct. Mater. 2014, 24, 3059-3066. (26) Chen, Y.; Zhu, Y.; Yang, D.; Luo, Q.; Yang, L.; Huang, Y.; Zhao, S.; Lu, Z. Asymmetrical Squaraines For High-Performance Small-Molecule Organic Solar Cells with a Short Circuit Current of over 12 Ma Cm− 2. Chem. Commun. 2015, 51, 6133-6136. (27) Sreejith, S.; Joseph, J.; Lin, M.; Menon, N. V.; Borah, P.; Ng, H. J.; Loong, Y. X.; Kang, Y.; Yu, S. W. K.; Zhao, Y. Near-Infrared Squaraine Dye Encapsulated Micelles for in Vivo Fluorescence and Photoacoustic Bimodal Imaging. ACS nano 2015, 9, 5695-5704. (28) McNamara, L. E.; Rill, T. A.; Huckaba, A. J.; Ganeshraj, V. G.; Gayton, J.; Nelson, R. A.; Sharpe, E. A.; Dass, A.; Hammer, N. I.; Delcamp, J. H. Indolizine–Squaraines: NIR Fluorescent Materials with Molecularly Engineered Stokes Shifts. Chem. Eur. J. 2017, 23, 12494-12501. (29) Jradi, F. M.; Kang, X.; O’Neil, D.; Pajares, G.; Getmanenko, Y. A.; Szymanski, P.; Parker, T. C.; El-Sayed, M. A.; Marder, S. R. Near-Infrared Asymmetrical Squaraine Sensitizers for Highly Efficient Dye Sensitized Solar Cells: The Effect of Π-Bridges and Anchoring Groups on Solar Cell Performance. Chem. Mater. 2015, 27, 2480-2487. (30) Paek, S.; Choi, H.; Jo, H.; Lee, K.; Song, K.; Siddiqui, S.; Sharma, G.; Ko, J. A New Unsymmetrical Near-IR Small Molecule with Squaraine Chromophore for Solution Processed Bulk Heterojunction Solar Cells. J. Mater. Chem. C 2015, 3, 7029-7037. (31) Yang, L.-N.; Sun, Z.-Z.; Li, Q. S.; Chen, S. L.; Li, Z. S.; Niehaus, T. A. Unsymmetrical Squaraine Dye Containing Dithieno [3, 2-b: 2′, 3′-d]pyrrole as a π-Spacer: A Potential Photosensitizer for Dye-sensitized Solar Cells. J. Power Sources 2014, 268, 137-145. (32) Wu, Y.; Huang, S.; Zeng, F.; Wang, J.; Yu, C.; Huang, J.; Xie, H.; Wu, S. A Ratiometric Fluorescent System for Carboxylesterase Detection with AIE dots as FRET Donors. Chem. Commun. 2015, 51, 12791-12794 (33) Qi, Y.; Huang, Y.; Li, B.; Zeng, F.; Wu, S. Real-time Monitoring of Endogenous Cysteine Levels in Vivo by Near-Infrared Turnon Fluorescent Probe with Large Stokes Shift. Anal. Chem. 2018, 90, 1014-1020. (34) Yu, C.; Li, X.; Zeng, F.; Zheng, F.; Wu, S. Carbon-dot-based Ratiometric Fluorescent Sensor for Detecting Hydrogen Sulfide in Aqueous Media and Inside Live Cells. Chem. Commun., 2013, 49, 403-405. (35) Sun, J.; Ye, B.; Xia, G.; Wang, H. A Multi-responsive Squaraine-based “Turn on” Fluorescent Chemosensor for Highly Sensitive Detection of Al3+, Zn2+ and Cd2+ in Aqueous Media and



Normal Subjects and Patients with Cancer and Other Diseases. Cancer 1958, 11, 283-291. (51) Monis, B.; Nachlas, M. M.; Seligman, A. M. Study of Leucine Aminopeptidase in Neoplastic and Inflammatory Tissues with A New Histochemical Method. Cancer 1959, 12, 601-608. (52) Iwahashi, T.; Okochi, E.; Ariyoshi, K.; Watabe, H.; Amann, E.; Mori, S.; Tsuruo, T.; Ono, K. Iwahashi, T.; Okochi, E.; Ariyoshi, K.; Watabe, H.; Amann, E.; Mori, S.; Tsuruo, T.; Ono, K. Specific Targeting and Killing Activities of Anti-P-Glycoprotein Monoclonal Antibody MRK16 Directed against Intrinsically Multidrug-resistant Human Colorectal Carcinoma Cell Lines in the Nude Mouse Model. Cancer Res. 1993, 53, 5475-5482. (53) Kelland, L. “Of mice and men”: Values and Liabilities of the Athymic Nude Mouse Model in Anticancer Drug Development. Eur. J. Cancer 2004, 40, 827-836. (54) Xie, X. S. Enzyme Kinetics, Past and Present. Science 2013, 342, 1457-1459. (55) Scornik, O.; Botbol, V. Bestatin as an Experimental Tool in Mammals. Curr. Drug Metab. 2001, 2, 67-85. (56) Chandra, D.; Ramana, K. V.; Wang, L.; Christensen, B. N.; Bhatnagar, A.; Srivastava, S. K. Inhibition of Fiber Cell Globulization and Hyperglycemia-Induced Lens Opacification by Aminopeptidase Inhibitor Bestatin. Invest. Ophthalmol. Vis. Sci. 2002, 43, 2285-2292. (57) Xu, Y.; Dresden, M. H. Leucine Aminopeptidase and Hatching of Schistosoma Mansoni Eggs. J. Parasitol. 1986, 507-511. (58) Burley, S. K.; David, P. R.; Lipscomb, W. N. Leucine Aminopeptidase: Bestatin Inhibition and a Model for EnzymeCatalyzed Peptide Hydrolysis. Proc. Natl. Acad. Sci. U. S. A 1991, 88, 6916-6920. (59) Gong, Q.; Shi, W.; Li, L.; Ma, H. Leucine Aminopeptidase May Contribute to the Intrinsic Resistance of Cancer Cells toward Cisplatin as Revealed by an Ultrasensitive Fluorescent Probe. Chem. Sci. 2016, 7, 788-792. (60) He, X.; Xu, Y.; Shi, W.; Ma, H. Ultrasensitive Detection Of Aminopeptidase N Activity in Urine and Cells with a Ratiometric Fluorescence Probe. Anal. Chem. 2017, 89, 3217-3221. (61) Zhou, Z.; Wang, F.; Yang, G.; Lu, C.; Nie, J.; Chen, Z.; Ren, J.; Sun, Q.; Zhao, C.; Zhu, W. H. A Ratiometric Fluorescent Probe for Monitoring Leucine Aminopeptidase in Living Cells and Zebrafish Model. Anal. Chem. 2017, 89, 11576-11582. (62) Zhang, W.; Liu, F.; Zhang, C.; Luo, J.-G.; Luo, J.; Yu, W.; Kong, L. Near-Infrared Fluorescent Probe with Remarkable Large Stokes Shift and Favorable Water Solubility for Real-Time Tracking Leucine Aminopeptidase in Living Cells and In Vivo. Anal. Chem. 2017, 89, 12319-12326. (63) David, N.A. The Pharmacology of Dimethyl Sulfoxide. Annu. Rev. pharmacol. 1972, 12, 353-374. (64) Antoine, D. J.; Dear, J. W.; Lewis, P. S.; Platt, V.; Coyle, J.; Masson, M.; Thanacoody, R. H.; Gray, A. J.; Webb, D. J.; Moggs, J. G. Mechanistic Biomarkers Provide Early and Sensitive Detection of Acetaminophen‐Induced Acute Liver Injury at First Presentation to Hospital. Hepatology 2013, 58, 777-787. (65) Salomone, F.; Barbagallo, I.; Puzzo, L.; Piazza, C.; Volti, G. L. Efficacy of Adipose Tissue-Mesenchymal Stem Cell Transplantation in Rats with Acetaminophen Liver Injury. Stem Cell. Res. 2013, 11, 1037-1044.

Its Biological Application. Sensors Actuators B: Chem. 2017, 249, 386-394. (36) Sun, W.; Guo, S.; Hu, C.; Fan, J.; Peng, X. Recent Development of Chemosensors Based on Cyanine Platforms. Chem. Rev. 2016, 116, 7768-7817. (37) Wang, G.; Xu, W.; Guo, Y.; Fu, N. Near-Infrared Squaraine Dye as A Selective Protein Sensor Based on Self-Assembly. Sensors Actuators B: Chem. 2017, 245, 932-937. (38) Xia, G.; Wang, H. Squaraine dyes: The Hierarchical Synthesis and Its Application in Optical Detection. J. Photoch. Photobio.C 2017, 31, 84-113. (39) Saikiran, M.; Sato, D.; Pandey, S. S.; Hayase, S.; Kato, T. Efficient Near Infrared Fluorescence Detection of Elastase Enzyme Using Peptide-Bound Unsymmetrical Squaraine Dye. Bioorg. Med. Chem. Lett. 2017, 27, 4024-4029. (40) Wu, N.; Lan, J.; Yan, L.; You, J. A Sensitive Colorimetric and Fluorescent Sensor Based on Imidazolium-Functionalized Squaraines for the Detection of GTP and Alkaline Phosphatase in Aqueous Solution. Chem. Commun. 2014, 50, 4438-4441. (41) Shimi, M.; Sankar, V.; Rahim, M. A.; Nitha, P.; Das, S.; Radhakrishnan, K.; Raghu, K. Novel Glycoconjugated Squaraine Dyes for Selective Optical Imaging of Cancer Cells. Chem. Commun. 2017, 53, 5433-5436. (42) Zheng, F.; Zeng, F.; Yu, C.; Hou, X.; Wu, S. A PEGylated Fluorescent Turn‐On Sensor for Detecting Fluoride Ions in Totally Aqueous Media and Its Imaging in Live Cells. Chem. Eur. J. 2013, 19, 936-942. (43) Fan, J.; Fang, G.; Zeng, F.; Wang, X.; Wu, S. Water‐ Dispersible Fullerene Aggregates as a Targeted Anticancer Prodrug with both Chemo‐and Photodynamic Therapeutic Actions. Small 2013, 9, 613-621. (44) Sunoqrot, S.; Bugno, J.; Lantvit, D.; Burdette, J. E.; Hong, S. Prolonged Blood Circulation and Enhanced Tumor Accumulation of Folate-Targeted Dendrimer-Polymer Hybrid Nanoparticles. J. Controlled Release 2014, 191, 115-122. (45) Qhattal, H. S. S.; Hye, T.; Alali, A.; Liu, X. Hyaluronan Polymer Length, Grafting Density, and Surface Poly(ethylene glycol) Coating Influence in Vivo Circulation and Tumor Targeting of Hyaluronan-Grafted Liposomes. ACS nano 2014, 8, 5423-5440. (46) Mericas, G.; Anagnostou, E.; Kakari, S. The Diagnostic Value of Serum Leucine Aminopeptidase. J. Clin. Pathol. 1964, 17, 52-55. (47) Banks, B. M.; Pineda, E. P.; Goldbarg, J. A.; Rutenburg, A. M. Clinical Value of Serum Leucine Aminopeptidase Determinations. New Engl. J. Med. 1960, 263, 1277-1281. (48) Salaspuro, M.; Sipponen, P.; Ikkala, E.; Kolho, L.; Makkonen, H.; Miettinen, T.; Räsänen, J.; Siurala, M. Clinical Correlations and Significance of Orceinpositivity in Chronic Active Hepatitis and Primary Biliary Cirrhosis. Ann. Clin. Res. 1976, 8, 206-215. (49) Cifaldi, L.; Romania, P.; Lorenzi, S.; Locatelli, F.; Fruci, D. Role of Endoplasmic Reticulum Aminopeptidases in Health and Disease: From Infection to Cancer. Int. J. Mol. Sci. 2012, 13, 83388352. (50) Goldbarg, J. A.; Rutenburg, A. M. The Colorimetric Determination of Leucine Aminopeptidase in Urine And Serum of


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